Controllable Transgene Expression Systems in Plants
One of the major problems in plant biotechnology is the achievement of reliable control over transgene expression. Tight control over gene expression in plants is essential if a downstream product of transgene expression is growth inhibitory or toxic, like for example, biodegradable plastics (Nawrath, Poirier & Somerville, 1994, Proc. Natl. Acad. Sci., 91, 12760-12764; John & Keller, 1996, Proc. Nail. Acad. Sci., 93, 12768-12773; U.S. Pat. No. 6,103,956; U.S. Pat. No. 5,650,555) or protein toxins (U.S. Pat. No. 6,140,075). Existing technologies for controlling gene expression in plants, are usually based on tissue-specific and inducible promoters and practically all of them suffer from a basal expression activity even when uninduced, i.e. they are “leaky”. Tissue-specific promoters (U.S. Pat. No. 5,955,361; WO09828431) represent a powerful tool but their use is restricted to very specific areas of applications, e.g. for producing sterile plants (WO9839462) or expressing genes of interest in seeds (WO00068388; U.S. Pat. No. 5,608,152). Inducible promoters can be divided into two categories according to their induction conditions: those induced by abiotic factors (temperature, light, chemical substances) and those that can be induced by biotic factors, for example, pathogen or pest attack. Examples of the first category are heat-inducible (U.S. Pat. No. 5,187,287) and cold-inducible (U.S. Pat. No. 5,847,102) promoters, a copper-inducible system (Melt et al., 1993, Proc. Natl. Acad. Sci., 90, 4567-4571), steroid-inducible systems (Aoyama & Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998, Plant J., 14, 247-257; U.S. Pat. No. 6,063,985), an ethanol-inducible system (Caddick et al., 1997, Nature Biotech., 16, 177-180; WO09321334), and a tetracycline-inducible system (Weinmann et al., 1994, Plant J., 5, 559-569). One of the latest developments in the area of chemically inducible systems for plants is a chimaeric promoter that can be switched on by the glucocorticoid dexamethasone and switched off by tetracycline (Bohner et al., 1999, Plant J., 19, 87-95). For a review on chemically inducible systems see: Zuo & Chua, (2000, Current Opin. Biotechnol., 11, 146-151). Other examples of inducible promoters are promoters which control the expression of pathogenesis-related (PR) genes in plants. These promoters can be induced by treatment of a plant with salicylic acid, an important component of plant signaling pathways in response to pathogen attack, or other chemical compounds (benzo-1,2,3-thiadiazole or isonicotinic acid) which are capable of triggering PR gene expression (U.S. Pat. No. 5,942,662).
There are reports of controllable transgene expression systems using viral RNA/RNA polymerase provided by viral infection (for example, see U.S. Pat. No. 6,093,554; U.S. Pat. No. 5,919,705). In these systems, a recombinant plant DNA sequence includes the nucleotide sequences from the viral genome recognized by viral RNA/RNA polymerase. The effectiveness of these systems is limited because of the low ability of viral polymerases to provide functions in trans, and their inability to control processes other than RNA amplification. Another way is to trigger a process of interest in a transgenic plant by using a genetically-modified virus which provides a heterologous nucleic acid encoding a switch for a biochemical process in a genetically-modified plant (WO02068664).
The systems described above are of significant interest as opportunities of obtaining desired patterns of transgene expression, but they do not allow tight control over the expression patterns, as the inducing agents (copper) or their analogs (brassinosteroids in case of steroid-controllable system) can be present in plant tissues at levels sufficient to cause residual expression. Additionally, the use of antibiotics and steroids as chemical inducers is not desirable or economically unfeasible for large-scale applications. When using promoters of PR genes or viral RNA/RNA polymerases as control means for transgenes, the requirements of tight control over transgene expression are also not fulfilled, as casual pathogen infection or stress can cause expression. Tissue- or organ-specific promoters are restricted to very narrow areas of application, since they confine expression to a specific organ or stage of plant development, but do not allow the transgene to be switched on at will. Recombinant viral switches as described in WO02/068664 address all these problems, but do not guarantee tight environmental safety requirements, as the heterologous nucleic acid in the viral vector can recombine.
There is abundant literature including patent applications which describe the design of virus resistant plants by the expression of viral genes or mutated forms of viral RNA (e.g. U.S. Pat. No. 5,792,926; U.S. Pat. No. 6,040,496). However, there is an environmental risk associated with the use of such plants due to the possibility of forming novel viruses by recombination between the challenging virus and transgenic viral RNA or DNA (Adair & Kearney, 2000, Arch. Virol, 145, 1867-1883).
Hooykaas and colleagues (2000, Science, 290, 979-982; WO01/89283) described the use of a translational fusion of Cre recombinase with vir gene fragments for Agrobacterium-mediated recombinase translocation into plant cells. Cre-mediated in planta recombination events resulted in a selectable phenotype. The translocation of Cre recombinase is the first use of a translocated protein as a switch to trigger a process of interest in plant cells. However, despite the translocation is not necessarily accompanied by DNA transfer, this approach does not guarantee high level safety, as the phytopathogenic genetically-modified microorganism (Agrobacterium) possesses a complete coding sequence of the switching protein Cre recombinase. Further, the process of interest can only be triggered in cells that receive the switching protein. If large ensembles of cell are to be treated, the ratio of cells receiving the switching protein to the total number of cells becomes very small. The method of Hooykaas can therefore not be applied to entire plants. Instead, its usefulness is limited to cells in tissue culture or cell culture. Further, as this method is limited to cell cultures (laboratory scale), environmental safety concerns as in large-scale or farm field applications do not arise.
It is therefore an object of the invention to provide an environmentally safe method of switching on a cellular process of interest in plants, whereby the cellular process may be selectively switched on at any predetermined time. It is another object of this invention to provide a method of switching on a cellular process of interest in entire plants. It is another object of this invention to provide a method for producing a product in a transgenic plant, wherein the production of the product may be selectively switched on after the plant has grown to a desired stage, whereby the process is environmentally safe in that genetic material necessary for said cellular process and genetic material coding for the control function are not spread in the environment together.